Extracellular proteolysis is an essential mechanism that mediates tissue homeostasis in the body. The matrix metalloproteinases (MMPs) are key players in the molecular communication between normal breast epithelium and its microenvironment during development. In breast cancer, the MMP profile is often severely altered, leading to unregulated tumor growth, tissue remodeling, inflammation, tissue invasion and metastasis. We have studied the role of MMPs and the extracellular matrix (ECM) in mammary development and cancer for 20 years. Our studies have led to an understanding of the morphogenetic and tumor-promoting functions of MMPs, as well as to basic cellular and molecular mechanisms underlying branching morphogenesis, collective invasion and stem cell function. Using an integrated series of assays with genetically modified mice and mammary cells, organotypic culture models, in vivo transplantation systems and imaging technology, we have gained significant insights into molecular mechanisms underlying altered epithelial cell function. One aspect of MMP function that is currently reorienting our thinking is the discovery of non-proteolytic functions of MMPs. We have found that MMP3 regulates Wnt signaling and alters mammary stem cells. Concentrating on MMP3, and to a lesser extent, MMP9 and MMP2, we will use structure-function studies in vivo to determine non-proteolytic and proteolytic functions, new accurate methods for evaluating mammary adult stem cells in vivo and in culture. Given our novel insights indicating that, depending on the circumstances, MMPs may function independently of their proteolytic activity, we now propose to (1) further analyze the mechanisms by which non-proteolytic functions of MMPs regulate mammary epithelial morphogenesis; (2) determine how mammary stem cells are regulated by MMPs; and (3) evaluate the role that extracellular matrix and MMP signaling in the tumor microenvironment play in regulating mammary neoplastic progression and metastasis. Understanding the molecular mechanisms of this complex interplay between malignant cancer cells and the surrounding non- malignant stroma represents one of the major challenges in cancer research, which once understood, will foster new approaches to cancer therapy,